The reconstituted 'humanized liver' in TK-NOG mice is mature and functional

Abstract

To overcome the limitations of existing models, we developed a novel experimental in vivo platform for replacing mouse liver with functioning human liver tissue. To do this, a herpes simplex virus type 1 thymidine kinase (HSVtk) transgene was expressed within the liver of highly immunodeficient NOG mice (TK-NOG). Mouse liver cells expressing this transgene were ablated after a brief exposure to a non-toxic dose of ganciclovir (GCV), and transplanted human liver cells are stably maintained within the liver (humanized TK-NOG) without exogenous drug. The reconstituted liver was shown to be a mature and functioning "human organ" that had zonal position-specific enzyme expression and a global gene expression pattern representative of mature human liver; and could generate a human-specific profile of drug metabolism. The 'humanized liver' could be stably maintained in these mice with a high level of synthetic function for a prolonged period (8 months). This novel in vivo system provides an optimized platform for studying human liver physiology, including drug metabolism, toxicology, or liver regeneration.

Figure 1

(A) Reconstituted ‘humanized liver’ in TK-NOG mouse. Immunohistochemical staining of sections obtained from TK-009-18 liver using h-CK8/18 antibody. The RI (%) for each section is indicated. Scale bar: 1 mm. An enlarged view of the boxed area is shown in the inset right. Scale bars: 100 μm. P, portal tract; C, central vein. (B) The human albumin (hAlb) level in the plasma of 4 TK-NOG mice and their body weight (BW) was measured over a 9-month period after GCV conditioning and human liver cell transplantation. (C) Immunoblot analyses of sera from 3 humanized TK-NOG mice with antibodies for specific for human albumin (hAlb), complement C3 proteins (hC3), transferrin (hTF), and for human and mouse ceruloplasmin (h/mCp). The RI of these mice (TK12-14, 6-6-10, and 2-4-7) mice were approximately 10, 30 and 85%, respectively. (D) Comparison of total protein (TP), total cholesterol (TCHO) albumin (ALB), ammonia (NH₃), and serum blood urea nitrogen (BUN) levels between the control NOG and humanized TK-NOG mice. The hAlb level in all of the humanized mice is >3 mg/mL. Open rhomboid, control NOG mice; filled rhomboid, humanized TK-NOG mice. (E) H&E and PAS staining of kidney obtained from a humanized TK-NOG mouse (hAlb >5.0 mg/mL). Scale bars, 100 μm.

Figure 2

(A) Histology and immunohistochemistry of the liver of humanized TK-NOG mice. Serial liver sections were stained for H&E, HLA, ASGR1, h-albumin, PAS, and h-CK8/18. Scale bars, 100 μm. (B) The position-specific pattern of GS expression within the liver lobule. H&E and immunohistochemical staining with anti-GS antibody in liver sections obtained from humanized TK-NOG mice 14 weeks after transplantation of human liver cells. The ‘Mouse’ and ‘Human’ hepatocyte boundary is indicated by a dashed line in the images on the left. Liver sections from control NOG or human livery sections that were stained with the anti-GS antibody are shown on the right. Scale bars: 100 μm. P, portal tract; C, central vein.

Figure 3

(A) Gene expression within the reconstituted ‘humanized liver’ of TK-NOG mice. Global gene expression profiles within a fully humanized TK-NOG liver (RI of >85%) and donor human liver cells were compared by microarray analysis. The blue and black triangles indicate ‘absent or marginal’ and ‘present’ detection calls, respectively. The red circles indicate the probe sets for mRNAs related to drug metabolism. (B) The relative expression of 26 human drug metabolism related mRNAs in four independent ‘humanized’ TK-NOG livers and in donor human liver cells (nHeps) was assessed by qPCR. Each bar represents the average of 3 independent determinations and the standard error is shown. (C) Human (CYP2D6)-specific drug biotransformation in humanized TK-NOG mice. The serum concentration of DEB (left panel) and 4-OH DEB (right panel) in control NOG (n = 9) and humanized TK-NOG (n = 6) mice were measured 0, 0.5, 1, 2, 4 and 7 hrs after administration of a single oral dose of DEB (2 mg/kg). Each data point represents the average ± SD of 6-9 independent mice tested. (D) Urine samples were collected between 0 and 7 hrs after DEB administration. The % ratio of 4-OH DEB/DEB excreted into urine within 7 hr was compared between control NOG (n = 9) and humanized TK-NOG mice (n = 6). By all of these measures, humanized TK-NOG mice had significantly increased amounts of 4-OH DEB in the plasma and higher 4-OH DEB excretion than control NOG mice.